Polymorphisms of SLC22A9 (Hoat7) in Korean Females with Osteoporosis

Korean J Physiol Pharmacol Vol 19: 319－325, July, 2015 pISSN 1226-4512 http://dx.doi.org/10.4196/kjpp.2015.19.4.319 eISSN 2093-3827

Polymorphisms of SLC22A9 (hOAT7) in Korean Females with Osteoporosis

Seong Kyu Ahn1, Chang Kook Suh2, and Seok Ho Cha1

Departments of 1Tropical Medicine and Parasitology, 2Physiology and Biophysics, College of Medicine, Inha University, Incheon 400-712, Korea

Among solute carrier proteins, the organic anion transporters (OATs) play an important role for the elimination or reabsorption of endogenous and exogenous negatively charged anionic compounds. Among OATs, SLC22A9 (hOAT7) transports estrone sulfate with high affinity. The net decrease of estrogen, especially in post-menopausal women induces rapid bone loss. The present study was performed to search the SN P within exon regions of SLC22A9 in Korean females with osteoporosis. Fifty healthy controls and 50 osteoporosis patients were screened for the genetic polymorphism in the coding region of SLC22A9 using GC-clamped PCR and denaturing gradient gel electrophoresis (DGGE). Six SNPs were found on the SLC22A9 gene from Korean women with/without osteoporosis. The SNPs were located as follows: two SN Ps in the osteoporosis group (A645G and T1277C), three SN Ps in the control group (G1449T, C1467T and C1487T) and one SNP in both the osteoporosis and control groups (G767A). The G767A, T1277C and C1487T SNPs result in an amino acid substitution, from synonymous vs nonsynonymous substitution arginine to glutamine (R256Q), phenylalanine to serine (F426S) and proline to leucine (P496L), respectively. The Km values and Vmax of the wild type, R256Q, P496L and F426S were 8.84, 8.87, 9.83 and 12.74 μ M, and 1.97, 1.96, 2.06 and 1.55 pmol/oocyte/h, respectively. The present study demonstrates that the SLC22A9 variant F426S is causing inter-individual variation that is leading to the differences in transport of the steroid sulfate conjugate (estrone sulfate) and, therefore this could be used as a marker for certain disease including osteoporosis.

Key W ords: SLC22A9, Polymorphism, Osteoporosis, Estrone sulfate, Denaturing gradient gel electrophoresis, GC-clamp

INTRODUCTION of estrogen [2]. The estrogen deficiency also induces an im- balance in bone remodeling, accelerates the loss of bone Bone is a very complicated tissue in which formation and mass increasing fracture risk [3,4]. Therefore, estrogen defi- resorption occur by a bone remodeling process. The loss of ciency can be characterized by high bone turnover with en- bone mass occurs in osteoporosis. Due to low bone mass, hanced osteoclastic bone resorption. The important factor there is increased bone fracture of hip, shoulder, wrist and in determining bone density and bone turnover in humans spine. Estrogen is the key hormone for maintaining bone is circulating estradiol [5-9]. The most abundant estrogen mass. It has been thought that estrogen deficiency is the precursor in circulation is the sulfate conjugate form of es- major cause of age-related bone loss in humans [1]. Oste- trogen such as estrone sulfate. The amount of this con- oporosis is initiated by the failure to acquire the optimal jugated form of estrogen is several fold higher than that peak of bone mass during growth and/or to maintain bone of unconjugated estrogens [10-12]. In addition, the estrone mass in later years [2]. The rapid loss of bone mass occurs sulfate is well known as the substrate of organic solute car- in post-menopausal women, mainly due to the net decrease riers such as SLC22A8 (hOAT3), SLC22A9 (hOAT7) and SLC22A11 (hOAT4) [13-15]. The single non-synonymous SNPs (nsSNPs) contribute Received January 21, 2015, Revised March 5, 2015, Accepted March 5, 2015 commonly to the functional diversity on encoded proteins in humans. It appears that nsSNP often lead to unexpected Corresponding to: Seok Ho Cha, Department of Tropical Medicine responses to drugs and changes in the susceptibility to dis- and Parasitology, College of Medicine, Inha University, 7-241 Shin- ease [16]. A number of reports indicated that nsSNPs affect heung-dong, Jung-gu, Incheon 400-712, Korea. (Tel) 82-32-890-0957, the functional roles of certain proteins in gene regulation (Fax) 82-32-890-0957, (E-mail) shcha@ inha.ac.kr by altering DNA and transcription factor binding and/or by affecting signal transduction [17-19]. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work ABBREVIATIONS: SLC, solute carrier; OAT, organic anion trans- is properly cited. porter; DGGE, denaturing gradient gel electrophoresis; SNP, single Copyright ⓒ Korean J Physiol Pharmacol. nucleotide polymorphism.

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In this report we screened SNPs of SLC22A9 in two lated genomic DNA, 0.5 mM of dNTP, 10 pmol of each pri- groups of Korean women: one group had osteoporosis and mer and 1 unit of DNA taq polymerase. Amplification was the second was a normal control group. We then performed performed using a Takara thermocycler (TP-3000) and the a functional analysis of the nsSNP from the osteoporosis PCR cycling conditions were as follows: an initial denatura- group by expression in Xenopus oocytes. tion at 94oC for 2 min, followed by 30 or 35 cycles of denaturation at 94oC for 1 min, annealing for 1 min at 55oC to 60oC, and elongation at 72oC for 1 min. The last cycle was METHODS followed by an additional elongation step of 72oC for 10 min. For optimal DGGE analysis, amplification was followed by Materials a heteroduplexing step, which includes denaturation at 98oC for 10 min, renaturation for 30 min at the annealing Taq DNA polymerase and polymerase chain reaction temperature of amplification, and extension at 37oC for 10 (PCR) primers were purchased from ELPIS BIO (Daejeon, min. The amplified products were checked by electro- Korea) and Bioneer (Daejeon, Korea), respectively. Restric- phoresis using 1.5 μl (about 1% of total volume) of each tion enzymes, oligo (dT), LA Taq DNA polymerase and product in a 1% agarose gel stained with 1 μg/ml of ethi- dNTPs were bought from TaKaRa Korea Biomedical Co. dium bromide solution. The primer sequences and anneal- (Seoul, Korea), and rNTP and m7G(5')ppp(5')G Cap ana- ing temperature are shown in Table 1. logue for synthesis of cRNA were purchased from Amer- sham Pharmarcia Biotech (Sunnyvale, CA, USA). T7 RNA Denaturing gradient gel electrophoresis (DGGE) polymerase was bought from Stratagene Cloning Systems Inc. (La Jolla, CA, USA) and RNasin (RNA inhibitor) was DGGE was performed using a V20-HCDC unit (www. purchased from Promega (Madison, WI, USA). The DNA scie-plas.com). PCR products were loaded onto a 230×250 blood mini kit was purchased from Qiagen (Hilden, mm, 0.5 mm-thick polyacrylamide gel (acrylamide: bisacry- Germany). [3H]estrone sulfate (53 Ci/mmol) was purchased lamide, 37.5:1) containing linear denaturing gradients (100% from NEN Life Science Products, Inc. All other chemicals UF=7 M urea/40% deionized formamide) [22,23]. The per- utilized in this study were insured for the highest purity centage of polyacrylamide was 10% and UF solution gra- available from commercial sources. dient was from 10% to 70%. Electrophoresis was performed in TAE buffer (40 mM Tris, 20 mM Acetic acid and 1 mM Patients EDTA) at 250 V for 3.5 h at 60oC. The gels were stained with ethidium bromide solution (1 μg/ml) for 10 min and Blood samples were taken from 50 osteoporosis patients photographed under an UV transilluminator. and 50 normal control subjects, which were approved by the Samsung Hospital Institutional Review Board (Cheil DNA sequencing and variant confirmation General Hospital & Women’s Healthcare Center IRB). All subjects involved in the present study were age matched Samples showing aberrant DGGE bending patterns were (40 to 65 years of age). The blood samples were kindly sup- used in PCR with non-GC clamp primers prior or after 80 plied from Dr. Ki Ok Han and also employed previous re- base pairs of the designated site. Samples showing addi- search [20]. tional heteroduplex bands were subjected to sequencing analysis for the exact determination of the sequence PCR reaction variant.

Genomic DNAs were extracted and used as template for Mutant construction the PCR reaction. The PCR primers were approximately 18 mers and were before/after each exon with the GC clamp The wild type SLC22A9 cDNA was obtained by screening on the forward or reverse primer [21]. Each PCR reaction a human kidney cDNA library and subcloned to pcDNA mixture (final volume of 12.5 μl) contained 50 ng of iso- 3.1(+). In order to prepare mutants SLC22A9, a modified

Table 1. Oligonucleotide primers for PCR amplification of the individual exons of the SLC22A9 gene

Exon Primer sequence Annealing Size Cycle No. Forward Reverse Temp. (°C) (bp)

1 Clamp-ATC AAC TGT TCA ACC TCA ACG AGA ACA GAG CCT CTT AC 60 30 490 2 TGG CTT CCT TCT CTT CCA G Clamp-TGT TCC ATA CAC ACT CAC 60 30 191 3 Clamp-ACC TGT TTC TGT TTC TCA G TCC AAA GTG TTG GCT TAC 60 35 242 4 Clamp-ATC TGT TTT TTC TTC CAG AGA AGA AAC AAA CTC ATA C 57 35 256 5 Clamp-TTC TCT TCC TTG GTC AG AGC TCC CAT CCA GCT CAC 60 30 209 6 Clamp-TTT CTG CTA TTG TTG AAG ACA CTG CAT GAA GCT TAC 60 30 205 7 Clamp-TCC TTT TTA ATC ATC ACA G ACC TGT GAT CTT TTC TCA C 60 30 303 8 Clamp-TGT TCT TCC TTT CTC CAG AGG GCA TAC TTA AGG TTC TGT AC 60 30 200 9 Clamp-TGC TGT TTC CAC TCA AAG ACC GGG CTA TTT ACT CAC 60 30 290 10 TCT TTG TAT TTG TTC TAG Clamp-CCT TGT TAT TTA TTT TC 55 35 227

Clamp sequence: CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC G+TAA TAA TAA T. SNPs of SLC22A9 in Osteoporosis Patients 321

Table 2. Oligonucleotide primers for mutant construct preparation of the SLC22A9 gene

Mutant Primer name Sequence (5’→3’)

G767A ApaI forward aat gac act ggg gcc ctc agc c PvuII reverse (mutation) acc agc tgg agg ata tgc cag tct Tga atg gc T1277C NdeI forward aca tgc cca aca tat gta aaa gga tct ccc reverse-1 (mutation) aaa cct cac gca gcg tct gca ttt ctt gtg gca caG ata tga tgg cca gaa ggc revers-2 att ggc aag agc aga cgc tcc taa gcc cag tgt tgc caa aac ctc acg c XmnI reverse-3 tgg gaa tta ctt cat ttc cat ggg caa aag caa ggg tat tgg caa gag cag acg c C1487T XmnI forward-1 aat gaa gta att ccc acc ata atc agg gca aga gct atg ggg atc aat gca acc forward-2 ggg atc aat gca acc ttt gct aat ata gca gga gcc ctg gct ccc ctc atg atg atc c forward-3 (mutation) tcc cct cat gat gat cct aag tgt gta ttc tcc acT cct gcc ctg gat cat cta tgg BamHI reverse ttc cac tct cgg atc ctc ttg ctt tgg

Restriction enzyme sites and mutated nucleotide were denoted by underline and bold capital letters, respectively.

PCR was performed using primer sets containing both mutation and restriction enzyme sites (Table 2). PCR was se- quentially performed for 12 cycles with the first primer (0.5 μM, 1 μl) and 12 cycles with the second primer (0.5 μM, 1 μl) and finally 35 cycles with the third primer (10 μM, 1 μl). The PCR product was sub-cloned using the TA cloning vector (Invitrogen) and confirmed its nucleotide sequences. The mutant constructs were prepared by substituting the mutated product into the wild type cDNA of SLC22A9 using the appropriate restriction enzyme digested cDNA. cRNA synthesis and uptake experiments using Xenopus laevis oocytes

cRNA synthesis and uptake experiments were performed as described previously [24]. The capped cRNAs were syn- Fig. 1. The exon-intron organization of the SLC22A9 genome and thesized in vitro with T7 polymerase from plasmid cDNAs its relationship to the SLC22A9 cDNA. Gray boxes represent the linearized with Xho I. Defolliculated oocytes were injected protein-coding regions of the exons. with 25 ng of the capped cRNAs and incubated in Barth’s solution (88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO3)2, 0.4 mM CaCl2, 0.8 mM MgSO4, 2.4 mM NaHCO3, and 10 mM of each exon, intron, and nucleotide sequence of the splice Hepes) containing 50 μg/ml gentamycin and 2.5 mM pyr- junction is shown in Table 3. The consensus sequences for uvate, pH 7.4, at 18oC. After incubation for 2∼3 days, up- RNA splicing (gt/ag) are found in the 5’ and 3’ termini for take experiments were performed at room temperature in each intron. The translation start codon (ATG) is present ND96 solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, in exon 1, and the translation termination codon (TAG) is 1 mM MgCl2, and 5 mM Hepes, pH7.4). The uptake reaction present in exon 10. was initiated by replacing ND96 solution containing various concentrations of radio-labeled estrone sulfate, which GC-clamp PCR, DGGE assay and screening for se- was terminated by the addition of sufficient ice-cold ND96 quence variation solution followed by five times washing after 1 h incubation. Oocytes were solubilized with 10% SDS and the radio- The DGGE method was used in order to determine the activity was determined by scintillation counting. SNP. The PCR conditions were tested over several annealing temperatures and cycle numbers using normal human chromosomal DNA. The typical patterns of each PCR prod- RESULTS ucts with synonymous and non-synonymous mutations found using GC-clamping primer sets separated on 1% Genomic organization of human SLC22A9 agarose gel are presented in Fig. 2A. The representative results of DGGE separated on acrylamide gel using several Using the SLC22A9 cDNA nucleotide sequence, we could control samples are shown in Fig. 2B. The results of DGGE find the SLC22A9 genomic DNA was from the NCBI data- showed a single band in the SNP found in exons. base (gi|568815587:63369670-63410919 Homo sapiens chro- To determine the SNP in Korean osteoporosis patients, mosome 11, GRCh38 Primary Assembly). The gene, ∼41.3 50 blood samples were obtained from osteoporosis and nor- kb long, is located on chromosome 11q13.1. Using the clon- mal subjects and screened. When the osteoporosis and con- ed SLC22A9 with the reported genomic sequence, an align- trol subjects were analyzed, six subjects showed different ment of the nucleotide sequence was performed to de- electrophoretic patterns (Fig. 2C). In general, PCR products termine the exon-intron gene organization. The SLC22A9 were produced in accordance with the different number of gene consists of 9 introns and 10 exons (Fig. 1). The size bands indicating that a mutation may have occurred. 322 SK Ahn, et al

Table 3. Exon-intron boundaries of the SLC22A9 gene

Exon Intron Exon

No. Size (bp) 3’ junction 5’ junction Size (bp) No. 3’ junction 5’ junction No.

1670 …CGTGACTGAG gtaagaggct… 676 1 …tctcttccag TGGGATCTGG… 2 2104 …TATCAGACAG gtgatgtgtg… 2405 2 …tgtttctcag GTTTGGGAGA… 3 3155 …ATTATGTTAA gtaagccaac… 95 3 …tttcttccag TAGCCGAGTG… 4 4169 …TGACCTCAAG gtatgagttt… 1582 4 …ccttggtcag TTGGCTGCTA… 5 5124 …AACCCTGGAG gtgagctgga… 6390 5 …attgttgaag ATTTTGAAAT… 6 6108 …CCTTTACGAG gtaagcttca… 24230 6 …atcatcacag ATTTGCAAAG… 7 7201 …GTGCCACAAG gtgagaaaag… 1414 7 …ctttctccag AAATGCAGAC… 8 8109 …CCATAATCAG gtacagaacc… 455 8 …ccactcaaag GGCAAGAGCT… 9 9204 …AGAAAAATGA gtgagtaaat… 922 9 …tttgttctag GAGAAAAGAC… 10 10 458 …AAAAAAAAAA

Bold gt and ag were consensus sequences for RNA splicing.

Fig. 3. Transport activity of the SLC22A9 and its variants. (A) Concentration dependence of estrone sulfate mediated by wild type SLC22A9 and variants in Xenopus laevis oocytes. Defolliculated stage VI and V oocytes were injected with 25 ng/oocyte of wild-type and variants cRNAs, and incubated for 3 days in Barth’s solution at 18°C. After three days, [3H] estrone sulfate uptake experiment was performed using various concentrations (25 nM∼50 μM). Each Fig. 2. The typical band patterns of agarose or acrylamide gel point employs 8∼10 oocytes and represented mean±standard error electrophoresis. (A) Normal individual genomic DNA amplified of three independent experiments. (B) Eadie-Hofstee plot analysis using DGGE primer sets for the respective mutation found in exons for determination of Km and Vmax values. by duplex PCR. (B) The duplex PCR products from the same genomic DNAs were loaded on a polyacrylamide gel. (C) The aberrant band patterns and sequencing electropherograms of observed SLC22A9 SNPs in osteoporosis and normal subjects. The synonymous A645G, G1449T and C1467T were found in exon 3 and 9, respectively. The G767A, T1277C and C1487T were found in exon 4, 7 and 9, respectively. SNPs of SLC22A9 in Osteoporosis Patients 323

Among these six SNPs, two SNPs were found in osteopo- Numerous large-scale screening were carried out to iden- rosis patients (one in exon 3 and the other in exon 7), three tify genetic variations that affect disease susceptibility and SNPs were found in exon 9 of normal subject and one SNP drug response as well as to determine the variants con- was found in both osteoporosis patients and normal sub- tributing to alter the phenotype. We found three nsSNPs jects (exon 4). In order to further clarify this, nucleotide from 50 Korean women osteoporosis patients and normal sequencing was performed. As shown in Fig. 2C, the SNPs controls. Unfortunately, there is no information of any func- found from exon 5, 7 and 9 showed that G in the 767th nu- tional analysis with discovered nsSNP. Our functional anal- cleotide of the coding region was changed to A (arginine ysis showed the increased value in Km (wild type, 8.84 μM to glutameine), T in the 1277th nucleotide of the coding re- vs. F426S, 12.74 μM) and decreased value in Vmax (wild gion was changed to C (phenylalanine to serine) and C in type, 1.97 pmol/oocyte/h vs. F426S, 1.55 pmol/oocyte/h) in the 1487th nucleotide of the coding region was changed to estrone sulfate transport. The transporting activities of the T (proline to leucine), respectively. However, other SNPs other two nsSNPs (R256Q and P496L) are similar to those did not change the amino acid sequences. of wild type SLC22A9. In human, the functional properties of transport have Functional analysis of human SLC22A9 variant in been reported. With respect to substrate specificity, hOAT3 Xenopus oocytes (SLC22A8) and hOAT4 (SLC22A11) also transport estrone and dehyroepiandrosterone sulfate. The differences between In order to examine whether the SLC22A9 polymorphism hOAT3, hOAT4 and SLC22A9 are the expression pattern found in Korean osteoporosis patients affects the functional in organ. The expression of OAT3 was observed in brain, activity on substrate transport, we constructed mutant skeletal muscle and kidney [13]. The OAT4 message was cDNAs and expressed them into Xenopus oocytes. As shown observed in kidney and placenta [15]. In contrast, SLC22A9 in Fig. 3, F426S exhibited reduced uptake for [3H] estrone was expressed exclusively in liver [14]. The result of our sulfate compared with that of the wild-type SLC22A9. ongoing study using Saos-2 and MG-63 osteosarcoma cell R256Q and P496L did not show a statistically significant line showed the expression of SLC22A11 (hOAT4) and change in [3H] estrone sulfate compared with those of the SLC22A9 (hOAT7) by polymerase chain reaction (data not wild-type SLC22A9. According to the changes of concen- shown). From these results, it might be thought of as in- tration-dependent [3H] estrone sulfate in wild type and direct evidence of steroid sulfate conjugate transport in F426S, the Km values for wild-type and F426S were 0.7 bone. When we screened the SNPs of SLC22A8 using the and 1.2 μM, respectively, and the Vmax values for wild- same patient blood samples, no nsSNP was found. In the type and F426S were 1.8 and 0.47 pmol/oocyte/h, respec- case of SLC22A11 (hOAT4), we have found one nsSNP and tively (Fig. 3). have reported the functional change of transporting activity of the mutant (E278K) in independent osteoporosis patients [20]. In order to understand the characteristics of DISCUSSION SLC22A8, 9 and 11, the study using cells derived from primary cultured or immortalized cells was necessary. In the present study, the genetic feature of the SLC22A9 Several transporter proteins such as the multidrug re- was investigated in the Korean women osteoporosis pa- sistance associated protein [27,28], organic anion trans- tients and normal subjects. We identified six SNPs (A645G, porting polypeptide C [29,30], Na+-taurocholate cotranspor- G767A, T1277C, G1449T, C1467T and C1487T) of SLC22A9 ting polypeptide [31,32] and the breast cancer resistance coding region from the osteoporosis and control subjects. protein (BCRP/ABCG2) [33,34] also transport steroids or Totally 3082 SNPs are reported in the National Center for their sulfate conjugates and their nsSNPs also showed a Biotechnology Information (NCBI) data base. Among them, decrease of their substrate transporting abilities [35-38]. It 1,335 synonymous and non-synonymous SNPs are observed has also reported that nsSNPs showing altered activity in in coding regions. They consist of 68 missense mutations some transporters is co-related with certain disease [39,40]. and three stop codon gained mutation within the coding In addition, several reports revealed that some SNPs of region. Two nsSNP, T1277C (F426S) and C1487T (P496L) SLC22A8 and SLC22A11 are located in the promoter site of SLC22A9, ares not yet reported in the NCBI data base. [41]. NCBI data base also shows some upstream SNP. The We also verify the existence of a functional alteration in study on the regulatory effect of upstream SNP will be also F426S. necessary. Human SLC22A9, a sulfate conjugate transporter, trans- In conclusion, we reported for the first time the nsSNPs ports estrone- and dehydroepiandrosterone sulfate as pre- of SLC22A9 in Korean women with osteoporosis and veri- dominant substrates. 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